Microorganisms have many types involving viruses, bacteria, fungi and the like, and are ubiquitous in that a large number of microorganisms are present in the environment, surface of human body, surface of various cavities inside the human body and even within organs. Microorganisms can be divided into harmful microorganisms and beneficial microorganisms in terms of human health. Infection with microorganisms will mostly lead to a disease under a certain condition, but some beneficial microorganisms are requisite factors for maintaining health balance of the human body. With the advances in studies on life sciences, researchers have found that microorganisms that are mixed and concomitantly living in colonies at specific sites in the human body have relatively stable types in a certain proportion. Changes in the types and relative abundance among microorganisms may influence human health, and in certain cases are even main pathogenic factors. At the same time, the interactions present among microorganisms are not limited to interactions of microorganisms of the same species. More and more research results indicate that interactions are also present between different species of microorganisms, for example, between bacteria and viruses, between bacteria and fungi, between viruses and fungi, etc. Under some conditions, the interaction is just the reason causing the occurrence of diseases. Therefore, in recent years, scientists are paying increasing attentions to the system study taking microbial populations as one interactional organism, which is known by the name of Microbiome Study (Microbiom).
Prior to the appearance of large-scale sequencing techniques, the identification and classification among microorganisms mainly depend on traditional morphological, phenotypic, physiologic and biochemical classification, and these techniques have difficulty in large-scale batch detection, and are time-consuming, energy-consuming, and inaccurate in typing and quantification. With the rapid development of molecular biology, the current large-scale sequencing resolves this problem very well. A principle of the large-scale sequencing is achieved mainly based on features of genetic sequences of microorganisms.
Genetic sequences of the same species of microorganisms not only have conserved sequence regions that are essentially not varied or varied to a very low degree during evolution, but also have variable sequence regions expressed differentially among types. In nucleated organisms, ribosomal RNA (rRNA) and the corresponding encoding gene ribosomal DNA (rDNA) become very good regions, at the same time comprise two sequence regions and have appropriate length and size, so that they are suitable for sequence analysis under the frame of the prior art. At present, sequences of regions such as 5SrRNA/rDNA, 5.8SrRNA/rDNA, 16SrRNA/rDNA, 18SrRNA/rDNA, 23SrRNA/rDNA, and ITS (Internal transcribed space) are used more frequently. A particular principle consists in that universal primers are designed according to these conserved regions, thereby to obtain, by amplification, fragments of all the microorganisms of the same species in the sample collected, these fragments are sequenced to detect genetic sequences of a variable region between the two conserved universal primers, and difference of the sequences of these variable regions are analyzed so as to achieve classification of the microorganisms. The relative abundance among microorganisms can be known by detecting sequencing reads of different types of microorganisms.
The differences in genetic sequences among different species of microorganisms are relatively great, and conserved regions and sequences thereof for the sequencing detection are also frequently different. Different lengths of genetic sequences and amplified fragments are conditions restricting the amplification reaction. Therefore, at present, it is a relatively common practice to design an amplification kit, for the same species of microorganisms with the same universal primer, to carry out amplification and sequencing detection. Microorganisms with different universal primers are each amplified and sequenced separately, but amplification reactions and sequencing at different batches suffer from differences in amplification efficiency and other influencing factors across different batches. Thus the type and abundance detection of microorganisms can only be carried out in the same species, and at the same time the comparison cannot be carried out among different species of microorganisms colonized in the same spatial region.